Methods for modulating osteochondral development using bioelectrical stimulation

ABSTRACT

Compositions and methods are provided for modulating the growth, development and repair of bone, cartilage or other connective tissue. Devices and stimulus waveforms are provided to differentially modulate the behavior of osteoblasts, chondrocytes and other connective tissue cells to promote proliferation, differentiation, matrix formation or mineralization for in vitro or in vivo applications. Continuous-mode and pulse-burst-mode stimulation of cells with charge-balanced signals may be used. Bone, cartilage and other connective tissue growth is stimulated in part by nitric oxide release through electrical stimulation and may be modulated through co-administration of NO donors and NO synthase inhibitors. Bone, cartilage and other connective tissue growth is stimulated in part by release of BMP-2 and BMP-7 in response to electrical stimulation to promote differentiation of cells. The methods and devices described are useful in promoting repair of bone fractures, cartilage and connective tissue repair as well as for engineering tissue for transplantation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication 60/687,430 filed Jun. 3, 2005, U.S. provisional patentapplication 60/693,490 filed Jun. 23, 2005, U.S. provisional patentapplication 60/782,462 filed Mar. 15, 2006 and U.S. provisional patentapplication 60/790,128 filed Apr. 7, 2006.

BACKGROUND

Diseases and injuries associated with bone and cartilage have asignificant impact on the population. Approximately five million bonefractures occur annually in the United States alone. About 10% of thesehave delayed healing and of these, 150,000 to 200,000 nonunion fracturesoccur accompanied by loss of productivity and independence. In the caseof cartilage, severe and chronic forms of knee joint cartilage damagecan lead to greater deterioration of the joint cartilage and mayeventually lead to a total knee joint replacement. Approximately 200,000total knee replacement operations are performed annually and theartificial joint generally lasts only 10 to 15 years leading to similarlosses in productivity and independence.

Furthermore, the incidence of bone fractures is also expected to remainhigh in view of the incidence of osteoporosis as a major public healththreat for an estimated 44 million Americans. In the U.S. today, 10million individuals are estimated to already have the disease and almost34 million more are estimated to have low bone mass, placing them atincreased risk for osteoporosis. One in two women and one in four menover age 50 will have an osteoporosis-related fracture in theirremaining life. Osteoporosis is responsible for more than 1.5 millionfractures annually, including: 300,000 hip fractures; 700,000 vertebralfractures; 250,000 wrist fractures; and 300,000 fractures at othersites. The estimated national direct expenditures (hospitals and nursinghomes) for osteoporotic hip fractures were $18 Billion in 2002 (NationalOsteoporosis Foundation Annual Report, 2002).

Several treatments are currently available to treat recalcitrantfractures such as internal and external fixation, bone grafts or graftsubstitutes including demineralized bone matrix, platelet extracts andbone matrix protein, and biophysical stimulation such as mechanicalstrain applied through external fixators or ultrasound andelectromagnetic fields.

Similarly, typical treatment for cartilage injury, depending on lesionand symptom severity, are rest and other conservative treatments, minorarthroscopic surgery to clean up and smooth the surface of the damagedcartilage area, and other surgical procedures such as microfracture,drilling, and abrasion. All of these may provide symptomatic relief, butthe benefit is usually only temporary, especially if the person'spre-injury activity level is maintained.

Bone and other tissues such as cartilage respond to electrical signalsin a physiologically useful manner. Bioelectrical stimulation devicesapplied to non-unions and delayed unions were initiated in the 1960s andis now applied to bone and cartilage (Ciombor and Aaron, Foot AnkleClin. 2005, (4):579-93). Currently, a market and general acceptance oftheir role in clinical practice has been established. Less well-knownoutcomes attributed to bioelectrical stimulation are positive bonedensity changes (Tabrah, 1990), and prevention of osteoporosis (Chang,2003). A recent report offered adjunctive evidence that stimulation withpulsed electromagnetic field (PEMF) significantly accelerates boneformed during distraction osteogenesis (Fredericks, 2003).

At present, clinical use of electrotherapy for bone repair consists ofelectrodes implanted directly into the repair site or noninvasivecapacitive or inductive coupling. Direct current (DC) is applied via oneelectrode (cathode) placed in the tissue target at the site of bonerepair and the anode placed in soft tissues. DC currents of 5-100 μA aresufficient to stimulate osteogenesis. The capacitative couplingtechnique uses external skin electrodes placed on opposite sides of thefracture site. Sinusoidal waves of 20-200 Hz are typically employed toinduce 1-100 mV/cm electric fields in the repair site.

The inductive coupling (PEMF) technique induces a time-varying electricfield at the repair site by applying a time-varying magnetic field viaone or two electrical coils. The induced electric field acts as atriggering mechanism which modulates the normal process of molecularregulation of bone repair mediated by many growth factors. Bassett etal., were the first to report a PEMF signal could accelerate bone repairby 150% in a canine. Experimental models of bone repair show enhancedcell proliferation, calcification, and increased mechanical strengthwith DC currents. Such approaches also hold potential for cartilageinjuries.

Wounded tissue has an electrical potential relative to normal tissue.Electrical signals measured at wound sites, termed the “injurypotential” or “current of injury”, are DC (direct current) only,changing slowly with time. Bone fracture repair and nerve re-growthpotentials are typically faster than usual in the vicinity of a negativeelectrode but slower near a positive one, where in some cases tissueatrophy or necrosis may occur. For this reason, most recent research hasfocused on higher-frequency, more complex signals often with no net DCcomponent.

Unfortunately, most electrotherapeutic devices now available rely ondirect implantation of electrodes or entire electronic packages, or oninductive coupling through the skin using coils which generatetime-varying magnetic fields, thereby inducing weak eddy currents withinbody tissues which inefficiently provides the signal to tissues and thusin addition to bulky coils requires relatively large signal generatorsand battery packs. The need for surgery and biocompatible materials inthe one case, and excessive circuit complexity and input power in theother, has kept the price of most such apparatus relatively high, andhas also restricted the application of such devices to highly trainedpersonnel. There remains a need, therefore, for a versatile,cost-effective apparatus that can be used to provide bioelectricstimulation to differentially modulate the growth of osteochondraltissue to promote proper development and healing.

SUMMARY OF THE INVENTION

According to its major aspects and broadly stated, the present inventionprovides a method for modulating the growth or repair of, for examplebone tissue or cartilage, by administering an electrical signal todeveloping or damaged bone or cartilage tissue.

The present invention overcomes the shortcomings of prior art devicesand methods by enabling the delivery of bioelectrical signals optimizedto correspond to selected features of natural body signals resulting inaccelerated and more permanent healing. The signals described hereinconform to selected features of natural signals and consequently tissuessubjected to electrostimulation according to the present inventionundergo minimal physiological stress. In addition, the present inventionis non-invasive and cost-effective making it desirable for multipleapplications for personal and individual use. Furthermore, the presentmethods provide electrical stimulation where the electrical signalsclosely mimic selected characteristics of natural body signals. Thestimulated tissue is therefore subjected to minimal stress and growthand repair is greatly facilitated.

In contrast to conventional TENS-type devices, which are aimed atblocking pain impulses in the nervous system, the apparatus used withthe present methods operates at a stimulus level which is below thenormal human threshold level of pain sensation and as such, most usersdo not experience any sensation during treatment to repair or promotegrowth of bone.

The technology described herein uses a class of waveforms, some of whichare novel and other which are known to have positive biological effectson tissues when applied through inductive coils, but have not beendemonstrated to have positive biological effects through electrodesuntil now.

Although no commercial bioelectrical devices are currently approved forosteoporosis therapy, the present invention provides a promisingcandidate. As demonstrated herein, unique pulsed electromagnetic field(PEMF) wave patterns may be advantageously applied at both a macroscopiclevel (i.e. common bone fractures) as well as at microscopic levels(i.e. osteoblast development). Certain embodiments of the inventionmaximize the utility and application of desired PEMF waveforms: forexample, the spine, hip and/or wrist are the most common sites ofosteoporotic fracture, for such types of fractures the inventors providesimple, self-adhesive, skin contact electrode pads as electrotherapeuticdelivery vehicles. The use of such electrode pads results in theimprovement of bone mass at such key anatomical sites. At a microscopiclevel, the present inventors have identified specific PEMF waveforms andfrequencies that optimize osteoblast development. As described ingreater detail in the Examples (see Example 1) the inventors demonstratethat PEMF signals enhance osteoblast mineralization and matrixproduction, and that the signal confers structural features as well. Theinventors also show that other PEMF signals enhanced cell proliferationand accompanying increases in bone morphogenetic proteins (BMPs). Whileboth pulse-burst and continuous electrical signals may be used in thepresent invention, the administration of continuous rather thanpulse-burst signals provided the more pronounced effects onproliferation and mineralization.

The electrical signals of the present invention may be used to promotethe repair and growth of structural tissues such as bone and cartilage.However, such systems and methods need not be confined to use withintact organisms, since isolated cells or tissue cultures can also beaffected by electrotherapeutic waveforms (appropriate electrical stimulihave been observed to modify the rates of cell metabolism, secretion,and replication). Electrical signals are generally applicable to otherconnective tissues such as skin, ligaments, tendons, and the like. Theelectrical signals described herein may be used to stimulate othertissues to increase repair of the tissues and promote growth of tissuesfor transplantation purposes. Isolated skin cells, for example, might betreated with the devices and waveforms of the present invention in anappropriate growth medium to increase cell proliferation anddifferentiation in the preparation of tissue-cultured, autogenousskin-graft material. In a like manner, these bioelectric signals can beapplied directly to injured or diseased skin tissue to enhance healing.

Exogenous delivery of bioelectrical signals and progenitor cells such asbone marrow stromal cells-BMSCs to a fracture can lead to enhancedhealing and repair of recalcitrant fractures. Both of these factors(bioelectricity and cell recruitment) are, in fact, parts of the naturalhealing process. For these applications, electrical stimulation usingthe waveforms described herein can be applied immediately after injurywith an electrotherapy system. The electrotherapy system may belightweight, compact and portable. Both electrical stimulation anduniversal cell-based therapy can be applied within a few days afterinjury. Autologous cells may be added at time further after injury. Thepresent invention also provides methods to induce bone repair ordevelopment that regenerates natural tissues rather than scar tissue.

Accordingly, it is an object of the present invention to provide methodsfor modulating the proliferation and differentiation of bone tissue forfacilitation of bone repair and development by administering novelelectrical signals to bone tissue.

It is another object of the present invention to provide novel culturesystems comprising the use of PEMF for bone tissue engineering.

It is another object of the present invention to provide novel culturesystems of progenitor cells in combination with electrical stimulation.

It is another object of the present invention to provide kits for thegrowth of autologous and allogeneic tissues for transplantation into ahost in need thereof.

It is another object of the present invention to provide methods forelectrically stimulating uncommitted progenitor cells in vitro or invivo to induce proliferation or differentiation.

It is another object of the present invention to provide methods formodulating the growth of cartilage, bone or other connective tissue.

It is another object of the present invention to provide methods formodulating the expression and release of bone morphogenic proteins.

These and other objects, features, and advantages of the presentinvention will become apparent after review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a waveform used in stimulating bonefracture healing.

FIG. 2 a provides an illustration showing an effective electrical signalwaveform in pulse mode based on an inductive, coil waveform and adaptedfor skin application for promoting mineralization of bone.

FIG. 2 b provides an illustration showing an effective electrical signalwaveform in continuous mode for promoting mineralization of bone.

FIG. 3 a provides an illustration showing an effective electrical signalwaveform in pulse mode for promoting proliferation of bone cells.

FIG. 3 b provides an illustration showing an effective electrical signalwaveform in continuous mode for promoting proliferation of bone cells.

FIG. 4 provides an illustration showing an experimental lab chamber fordelivering current.

FIG. 5 provides a bar graph showing the changes in alkaline phosphatasein supernatant (left), and in membrane (right).

FIG. 6 provides a bar graph showing the changes in osteocalcin andcalcium deposits with signal “B”.

FIG. 7 provides a bar graph showing the increase in cell number measuredby DNA as a percentage of control±standard deviation for PEMF signalwaveforms in the presence and absence of L-NAME. L-NAME alone ispresented as an experimental control.

FIG. 8 provides schematics of setups for using a combination ofmechanical and electrical stimulation for in vitro applications.

DETAILED DESCRIPTION OF THE INVENTION

The following description includes the best presently contemplated modeof carrying out the invention. This description is made for the purposeof illustrating the general principles of the inventions and should notbe taken in a limiting sense. The text of the references mentionedherein are hereby incorporated in their entireties by reference,including U.S. Provisional Application Ser. Nos. 60/687,430, 60/693,490,60/782,462 and 60/790,128.

It should be understood that the present in vitro applications of theinvention described herein may also be extrapolated for in vivoapplications, therapies and the like. One of ordinary skill willappreciate that technology developed using reduced preparations and invitro models may ultimately be used for in vivo applications. Effectivevalues and ranges for electrical stimulation in vivo may be extrapolatedfrom dose-response curves derived from in vitro or animal model testsystems.

The present invention enables the delivery of bioelectrical signalsoptimized to correspond to selected characteristics of natural bodysignals resulting in accelerated and more permanent healing. The signalsdescribed herein uniquely conform to selected features of naturalsignals and consequently tissues subjected to electrostimulationaccording to the present invention undergo minimal physiological stress.In addition, the present invention is non-invasive and cost-effectivemaking it desirable for multiple applications for personal andindividual use.

Bone Remodeling

Bone is one of the most rigid tissues of the human body. As the maincomponent of the human skeleton, it not only supports muscularstructures but protects vital organs in the cranial and thoraciccavities. Bone is composed of intercellular calcified material (the boneextracellular matrix) and different cell types: osteoblasts, osteocytesand osteoclasts. The extracellular matrix is composed of organic andinorganic components. The organic component includes cells, collagens,proteoglycans, hyaluronan and other proteins, phospholipids and growthfactors. The rigidity of bone comes from the mineralized inorganiccomponent which is predominantly calcium and phosphorus crystallized inthe form of hydroxyapatite Ca₁₀(PO₄)₆(OH)₂. The combination of collagenand hydroxyapatite confers the hardness and stiffness characteristics ofbone.

Osteoblasts are derived from progenitor cells of mesenchymal origin andare localized at the surfaces next to emerging bone matrix and arrangedside-by-side. The primary function of osteoblasts is the elaboration anddevelopment of bone matrix and to play a role in matrix mineralization.Osteoblasts are called osteocytes when embedded in the lacunae of thebone matrix and adopt a slightly different morphology and retain contactwith other osteocytes. Osteoclasts are larger multinucleate cellscontaining receptors for calcitonin and integrin and other specializedstructural features. The primary function of osteoclasts is to resorbboth inorganic and organic components of calcified bone matrix.

Bone remodeling is the fundamental and highly integrated process ofresorption and formation of bone tissue that results in preciselybalanced skeletal mass with renewal of the mineralized matrix. Thisrenewable process is achieved without compromising the overallanatomical architecture of bones. This continuous process of internalturnover ensures that bone maintains a capacity for true regenerationand maintenance of bone integrity by continuous repairing ofmicrofractures and alterations in response to stress. The architectureand composition of the adult skeleton is in perpetually dynamicequilibrium. Remodeling also provides a means for release of calcium inresponse to homeostatic demands. Conditions that influence boneremodeling include mechanical stimuli such as immobilization orweightlessness, electric current or electromagnetic fields such ascapacitively coupled electric field or pulsed electromagnetic field,hormonal changes or in response to certain inflammatory diseases.

Bone remodeling occurs through orchestrated cycles of activity thatinclude activation, resorption, reversal, formation, and quiescencesteps. Activation is characterized by the existence of a thin layer oflining cells. Then circulating mononuclear cells of hematopoetic lineagebegin to migrate into the activation site and fuse together to formosteoclasts. Activation is followed by resorption where activeosteoclasts excavate a bony surface. This step typically lasts about 2-4weeks. Reversal occurs following resorption and continues for a periodof 9 days during this time inactive pre-osteoblasts are present in theresorption depressions. The next step is formation and takes about 3-4months. During this stage active osteoblasts refill the excavation site.The last phase of bone remodeling is quiescence where no remodelingactivity occurs until the beginning of the next remodeling cycle.Ideally the quantity of bone fill must equal the quantity resorbed withno loss of bone mass.

Waveforms

The present invention provides electrical signals and waveforms thatenable specific actions on biological tissues. Such waveforms areeffective for both in vivo and in vitro applications. Osteochondraltissues are shown herein to respond differently to markedly differentfrequencies and waveforms.

Of particular interest are signals comprising alternating rectangular orquasirectangular pulses having opposite polarities and unequal lengths,thereby forming rectangular, asymmetric pulse trains. Pulses of specificlengths have been theorized to activate specific cell biochemicalmechanisms, especially the binding of calcium or other small, mobile,charged species to receptors on the cell membrane, or their (usuallyslower) unbinding. The portions of such a train having oppositepolarities may balance to yield substantially a net zero charge, and thetrain may be either continuous or divided into pulse bursts separated byintervals of substantially zero signal. Stimuli administered inpulse-burst mode have similar actions to those administered ascontinuous trains, but their actions may differ in detail due to theability (theoretically) of charged species to unbind from receptorsduring the zero-signal periods, and required administration schedulesmay also differ.

FIG. 1 shows a schematic view of a base waveform 20 effective forstimulating bone and cartilage tissue, where a line 22 represents thewaveform in continuous mode, and line 24 represents the same waveform ona longer time scale in pulse-burst mode, levels 26 and 28 represent twodifferent characteristic values of voltage or current, and intervals 30,32, 34 and 36 represent the timing between specific transitions. Levels26 and 28 are usually selected so that, when averaged over a full cycleof the waveform, there is no net direct-current (D.C.) componentalthough levels 26 and 28 may be selected to result in a net positive ornet negative D.C. component if desired. In real-world applications,waveform such as 20 is typically modified in that all voltages orcurrents decay exponentially toward some intermediate level betweenlevels 26 and 28, with a decay time constant preferably longer thaninterval 34. The result is represented by a line 38. The waveformsdescribed herein generally have two signal components: a longercomponent shown as interval 30 and a shorter component shown as interval32 relative to each other.

Variation in the short and long signal component lengths confersspecific effects of a stimulated tissue. Pulse lengths of interest inthis invention may be defined as follows, in order of increasing length:

-   Length α: between 5 and 75 μsec in duration, preferably between 10    and 50 μsec in duration, more preferably between 20 and 35 μsec in    duration and most preferably about 28 μsec in duration.-   Length β: between 20 and 100 μsec in duration, preferably between 40    and 80 μsec in duration, more preferably between 50 and 70 μsec in    duration and most preferably about 60 μsec in duration.-   Length γ: between 100 and 1000 μsec in duration, preferably between    150 and 800 μsec in duration, more preferably between 180 and 500    μsec in duration and most preferably about 200 μsec in duration.-   Length δ: in excess of 1 millisecond in duration, preferably between    5 and 100 msec in duration, more preferably between 10 and 20 msec    in duration and most preferably about 13 msec in duration.

In a first embodiment the electrical signal has a shorter component oflength α and a longer component of length β: thus having, with the mostpreferable pulse lengths of each type (28 μsec and 60 μsecrespectively), a frequency of about 11.4 KHz. Signals comprised ofpulses alternately of length α and length β are referred to herein as“type A” signals and their waveforms as “type A” waveforms. An example a“type-A signal administered as a continuous pulse train is shown in FIG.2 a. Signals such as this are useful for promoting the proliferation ofa tissue sample or culture for a variety of biological or therapeuticapplications.

In pulse-burst mode, “type A” waveforms would be turned on in bursts ofabout 0.5 to 500 msec, preferably about 50 msec, with bursts repeated at0.1-10 Hz or preferably about 1 Hz. An example of this type of waveformis shown in FIG. 2 b.

In a second embodiment the electrical signal has a shorter component oflength α but a longer component of length γ: thus having, with the mostpreferable pulse lengths of each type (28 μsec and 200 μsecrespectively), a frequency of about 4.4 KHz. Signals comprised of pulsesalternately of length α and length γ are referred to herein as “type B”signals and their waveforms as “type B” waveforms. Such waveforms werepreviously described in U.S. patent application Ser. No. 10/875,801(publication no. 2004/0267333). An example of a “type-B” signaladministered as a continuous pulse train is shown in FIG. 3 a. Signalssuch as this are useful in pain relief and in promoting bone healing,and also stimulate the development of cancellous-bone-like structures inosteoblast cultures in vitro, with applications to the field of surgicalbone repair and grafting materials.

In pulse-burst mode, “type B” waveforms are turned on in bursts of about1 to 50 msec, preferably about 5 msec, with bursts repeated at 5-100 Hzor preferably about 15 Hz. An example of this type of waveform is shownin FIG. 3 b. This waveform is similar in shape and amplitude toeffective currents delivered by typical inductive (coil) electromagneticdevices that are commonly used in non-union bone stimulation productse.g. EBI MEDICA, INC.® (Parsippany, N.J.) and ORTHOFIX, INC.® (McKinney,Tex.).

In a third embodiment the electrical signal has a shorter component oflength β but a longer component of length γ: thus having, with the mostpreferable pulse lengths of each type (60 μsec and 200 μsecrespectively) a frequency of about 3.8 KHz. Signals comprised of pulsesalternately of length β and length γ are referred to herein as “type C”signals and their waveforms as “type C” waveforms. Signals such as thisare useful in promoting bone regeneration, maturation and calcification.

In pulse-burst mode, “type C” waveforms are turned on in bursts of about1 to 50 msec, preferably about 5 msec, with bursts repeated at 5-100 Hzor preferably about 15 Hz, much the same as “type B.” This waveform issimilar in shape and amplitude to effective currents delivered by othertypical inductive (coil) electromagnetic devices commonly used innon-union bone stimulation products, e.g. the ORTHOFIX, INC.® (McKinney,Tex.) PhysioStim Lite® which is designed to promote healing of spinalfusions.

In a fourth embodiment the electrical signal has a shorter component oflength γ and a longer component of length δ: thus having, with the mostpreferable pulse lengths of each type (200 μsec and 13 msecrespectively) a frequency of about 75 Hz. Signals comprised of pulsesalternately of length γ and length δ are referred to herein as “type D”signals and their waveforms as “type D” waveforms. Signals such as thisare useful especially in promoting cartilage healing and bonecalcification, and in treating or reversing osteoporosis andosteoarthritis. While broadly similar to that delivered throughelectrodes by the BIONICARE MEDICAL TECHNOLOGIES INC.® BIO-1000™, asshown in FIG. 3 of U.S. Pat. No. 5,273,033 which is here incorporated byreference, the “type D” signal differs substantially in wave shape (itis rectangular rather than exponential) and in the fact that it ispreferably charge-balanced.

In pulse-burst mode, “type D” waveforms are turned on in bursts of atleast 100 msec, preferably about 1 second, with bursts repeated atintervals of one second or more.

The signal intensity may also vary; indeed, more powerful signals oftengive no more benefit than weaker ones, and sometimes less. For a typicalsignal (such as the signal of FIG. 1), a peak effectiveness typicallyfalls somewhere between one and ten microamperes per square centimeter(μA/cm²), and a crossover point at about a hundred times this value.Beyond this point, the signal may slow healing or may itself causefurther injury.

Of particular relevance to the present methods are electrical signals orwaveforms, that run in continuous mode instead of burst mode. (Forexample FIGS. 2 a or 3 a). Continuously run signals have effects similarto those of pulse-burst signals, but may require different deliveryschedules to achieve similar results.

For the waveforms used with the methods of the present invention,typical applied average current densities are between 0.1 and 1000microamperes per square centimeter, preferably between 0.3 and 300microamperes per square centimeter, more preferably between 1 and 100microamperes per square centimeter, and most preferably about 10microamperes per square centimeter, resulting in voltage gradientsranging between 0.01 and 1000, 0.03 and 300, 0.1 and 100, and 1 and 10microamperes per centimeter, respectively, in typical body tissues. Theindividual nearly-square wave signal is asynchronous with a longpositive segment and a short negative segment or vice versa. Thepositive and negative portions balance to yield a zero net charge oroptionally may be charge imbalanced with an equalizing pulse at the endof the pulse to provide zero net charge balance over the waveform as awhole. These waveforms delivered by skin electrodes use continuousrectangular or approximately rectangular rather than sinusoidal orstrongly exponentially decaying waveforms. Other waveforms useful in themethods of the present invention are disclosed in published U.S. patentapplication Ser. No. 10/875,801 (publication no. 2004/0267333)incorporated herein by reference in its entirety.

The electrical signals described above may be administered to cells,biological tissues or individuals in need of treatment for intermittenttreatment intervals or continuously throughout the day. A treatmentinterval is defined herein as a time interval that a waveform isadministered in pulse or continuous mode. Treatment intervals may beabout 10 minutes to about 4 hours in duration, about 30 minutes to about2.5 hours in duration or about 1 hour in duration. Treatment intervalsmay occur between about 1 and 100 times per day. The duration andfrequency of treatment intervals may be adjusted for each case to obtainan effective amount of electrical stimulation to promote cellproliferation, cell differentiation, bone growth, development or repair.The parameters are adjusted to determine the most effective treatmentparameters.

Signals do not necessarily require long hours of duration in thetreatment interval although 24 hours administration may be used ifdesired. Typically, 30 minutes (repeated several times a day) isrequired for biological effectiveness. In vitro cell proliferation maybe measured by standard means such as cell counts, increases in nucleicacid or protein synthesis. Upregulation or down regulation of matrixproteins (collagen types I, III, and IV) as well as growth factors andcytokines (such as TGF-B, VEGF, SLPI, FN, MMPs) may also be measured(mRNA and protein synthesis). In vivo effects may be determined by rateof healing of an injury or measuring bone mass density. Other diagnosticmethods for proliferation, differentiation or mineralization of bonetissue will be readily apparent to one of ordinary skill.

In one embodiment, proliferation-promoting and differentiation-promotingsignals are used sequentially. This combination of waveforms is used toincrease the cell number and then promote differentiation of the cells.As an example, the sequential use of proliferation and differentiationsignals may be used to promote proliferation of osteoblasts and thendifferentiation of the osteoblasts into mineral producing osteocytesthat promote mineralization of bone or vice versa. For example, atreatment paradigm may be used where a proliferation-promoting A-typesignal is administered first to a cell population in vitro or ex vivofor hours, days or weeks and then the proliferation promoting signal isreplaced with a mineralization-promoting B-type signal for hours, daysor weeks until bone mineralization has been effected. The tissueproduced may then be transplanted for patient benefit. Both signals mayalso be applied simultaneously to promote both proliferation,differentiation and mineralization simultaneously.

The electric signals may be delivered by skin electrodes, orelectrochemical connection. Skin electrodes are available commerciallyin sizes such as 1½×12, 2×3½, and 2×2 inches that may be useful forapplication to the spine, hips, and arm, respectively. These reusableelectrodes are advantageous because they do not contain latex and havenot shown significant skin irritation. The reusable electrodes can beused multiple times; also reducing costs to the patient. Such electrodesmay include electrodes #214 (⅕″×13″), #220 (2″×2″) and #230 (2″×⅗″)(KOALATY PRODUCTS®, Tampa, Fla.) or electrodes #T2020 (2″×2″) and #T2030(2″×3.5″) (VERMED, INC®, Bellows Falls, Vt.).

There are multiple advantages of using skin electrodes instead ofelectromagnetic coils. Firstly, skin electrodes are more efficient. Withelectrodes, only the signal which will actually be sent into the bodymust be generated. With a coil, because of poor electromagnetic couplingwith the tissues, the signal put in must be many, many times strongerthan that desired in the tissues. This makes the required generatingcircuitry for electrodes potentially much simpler than for coils, whilerequiring much less power to operate. Secondly, skin electrodes are moreuser friendly. Skin electrodes have at most a few percent of the weightand bulk of coils needed to deliver equivalent signal levels. Similarly,because of better coupling efficiency the signal generators to driveelectrodes can be made much smaller and lighter than those for coils.After a short time, a wearer hardly notices they are there. Thirdly,skin electrodes are more economical. Unlike coils, which cost hundredsto thousands of dollars each, electrodes are “throw-away” itemstypically costing less than a dollar. Also, because of greaterefficiency and simplicity, the signal generators and batteries to drivethem can be small and inexpensive to manufacture compared with those forcoils. Fourthly, skin electrodes permit simpler battery construction andlonger battery life facilitating the ease and patient compliance ofusing the device. Lastly, skin electrodes are more versatile thanelectromagnetic coils. Coils must be built to match the geometriccharacteristics of body parts to which they will be applied, and eachmust be large enough to surround or enclose the part to be treated. Thismeans to “cover” the body there must be many, many different coil sizesand shapes, some of them quite large. With electrodes, on the otherhand, current distribution is determined by electrode placement only andreadily predictable throughout the volume between, so the body may be“covered” with just a few electrode types plus a list of well-chosenplacements.

Stimulation Systems

Also contemplated by the present invention are biological systems thatinclude cells and stimulators for delivering electrical signals tocells. Such cells may include, but are not limited to, precursor cellssuch as stem cells, uncommitted progenitors, committed progenitor cells,multipotent progenitors, pluripotent progenitors or cells at otherstages of differentiation. Such cells may be embryonic, fetal, or adultcells and may be harvested or isolated from autologous or allogeneicsources. In one embodiment proliferative cells are used althoughnon-proliferative cells may also be used in the methods describedherein. Such cells may be combined in vitro, for example in tissueculture, or in vivo for tissue engineering or tissue repairapplications. Transplanted stem cells may be selectively attracted tosites of injury or disease and then electrically stimulated to provideenhanced healing.

Stimulating cell cultures in accordance with the method and purpose ofthe present invention also requires a practical means of deliveringuniform waveforms simultaneously to many culture wells withoutdisturbing the incubation process or causing contamination. Devices areprovided herein for electrically stimulating cultures during incubationthat preferably contain six tissue culture wells connected as amulti-well system using specially designed capacitively coupled anodizedelectrode systems for signal administration. By using a ribbon cableattachment, leaks at the seal of the incubator are minimized maintainingthe controlled CO₂ environment for the cultures. A typical setup isshown, in partly schematic form, in FIG. 4.

In the setup shown for example in FIG. 4, six tissue culture wells 110 athrough 110 f are interconnected and each well includes electrodes 140at the chamber ends formed by two 15-mm and one 7.5-mm straight segmentsof wire, joined by hairpin bends and connected by a right-angle bend tothe central part 142 of the bridge 112. Seven such bridges are shown inFIG. 4. The electrodes 140 are sized to fit the end walls of a Lab-TekII slide chamber, which measures 18 by 48 millimeters internally with atypical 3-mm fill depth. The capacitance of such an electrode is about0.56 microfarad. Bridges 112 a and 112 g differ in having end-wellspirals 144 each containing about 15 cm of wire. The resultingcapacitance between the bridge wire 112 a or 112 g and the correspondingsilver electrode 104, is about 2.3 microfarads.

Bridges 112 a, 112 b and so forth, formed of solid, relatively inertmetal, connect chambers 100 a, 110 b and so forth electrically in seriesbetween end wells 106 a and 106 b. While six chambers 110 a through 110f, and seven bridges 112 a through 112 g, are shown here, any otherconvenient numbers “n” of chambers and “n+1” of bridges could be used.In addition, a plurality of such series-connected groups each comprisedof “n” chambers, “n+1” bridges and two end wells could be used with asingle signal source 100, using a signal distribution means such as aresistor network to divide the signal energy among the groups, as iswell known in the art of electronic signaling.

The total electrical impedance of the setup shown, with twelve chamberelectrode ends, two end-well spiral electrode 106 and six chambers asdescribed, is chiefly capacitive at 0.045 microfarad plus a resistivecomponent of about 10,000 ohms. A series resistor (not shown) connectedbetween signal source 100 and end well 106 a can both regulate theapplied current to a desired level and also “swamp out” the capacitivepart of the series reactance. For example, with a 1-Megohm resistor thefrequency response is uniform within ±3 dB from 5 Hz to 3 Mhz.

If desired, the signal energy distribution in a chamber may be measuredwith probes as shown in the magnified chamber 110 b. Probes 120, made ofany reasonably inert metal but preferably of 99.9% pure silver aselectrodes 104 a and 104 b, insulated except at their tips, and withthese tips set a known and fixed distance apart, are immersed in medium122 and moved into a succession of positions, preferably marking arectangular grid. The differential voltage at each position is read by adifferential amplifier 124, such as an Analog Devices AD522, and sent toan oscilloscope or other device, generally indicated by 126, for displayor recording. The results are conveniently represented as an array ofnumbers representing the ratio of signal intensity at each point to theoverall average, as shown at the bottom of FIG. 4 again for themagnified chamber 110 b. Alternatively, other means such as color-codingor three-dimensional graphing may be used.

The results are conveniently represented as an array of numbersrepresenting the ratio of signal intensity at each point to the overallaverage, as shown at the bottom of FIG. 4 again for the magnifiedchamber 110 b. Alternatively, other means such as color-coding orthree-dimensional graphing may be used.

As is shown by the grid in FIG. 4, the signal distribution withelectrodes placed at the narrow ends of a rectangular chamber istypically quite uniform save in the small regions immediately adjacentto the electrodes themselves. Uniformity also improves with time, eitherin medium or in plain saline, as cut or broken oxide heals. Theabove-average readings at lower left in FIG. 4, for example, may haveresulted from incompletely healed oxide at the cut wire end.

For convenience in handling, minimal medium evaporation and ease inmaintaining sterility, all of the chambers, bridges and end wells in agroup may conveniently be assembled on a rigid glass plate or othersterilizable carrier, and one of more of these plates once assembled maythen be enclosed in an outer container such as a rigid plastic box.

The present invention also provides novel stimulation devices fordelivering electrical signals in order to promote bone growth or repair.Specifically, novel passive electrode systems are provided fordelivering electrical signals. These electrode systems coupletime-varying electric signals for in vitro or in vivo applications; andreplace conventional electrolyte bridge technology for the delivery ofPEMF-type signals by induction in favor of a capacitive coupling. Theelectrode systems may be made of materials such as, but not limited to,anodized metals such as niobium, tantalum, titanium, zirconium,molybdenum, tungsten and vanadium. Aluminum and stainless steels sharethis property but to a much lesser degree, since they are slowlyattacked by solutions containing chloride ion. At usable frequencies,typically between about 5 Hz and 3 MHz and, with circuit refinement,from below about 1 Hz to in excess of about 30 MHz, DC current passageis negligible.

Niobium is one of several metals that is self-passivating therebyforming thin but very durable surface oxide layers when exposed tooxygen and moisture. This process can be controlled by anodization.Generally self-passivation makes reliable connection with other methodsdifficult, however the present design uniquely uses capacitive couplingto induce a current in the electrode and thereby avoids the difficultyof forming electrical connections with other metals. This electrodesystem provides negligible electrolysis and no physiologicallysignificant cytotoxicity and is also useful for in vivo applications.

The wires that are used with the electrode system of the presentinvention are “self-protecting,” forming thin, but very durable andtightly adhering surface layers of non-reactive oxides when exposed tomoisture or oxygen. The oxide so formed has a high dielectric constant,and the thickness of the oxide is substantially uniform and can beclosely controlled. The protective oxide coating allows the metal to actas a coupling capacitor for introducing alternating current (zero netcharge, or ZNC) electric signals to culture media with even distributionand negligible electrolysis.

A stimulator or other signal source, generally indicated by 100, isconnected through wires, clip leads or by any other convenient means 102to a pair of relatively inert metal electrodes 104 a and 104 b which areimmersed in electrically conductive fluid in end wells 106 a and 106 b.These provide an entry point for the signal to the assembly of culturechambers 110 a, 110 b and so forth to which it is to be applied. Fine(99.9% pure) silver is preferred for electrodes 104 a and 104 b, andsaline (sodium chloride solution) for the fluid in end wells 106 a and106 b, since in use a thin layer of silver chloride forms at theinterface and through a reversible electrochemical reaction facilitatesthe passage of electric current. Other metals and fluids, however, mayalso be used.

Bridges 112 a, 112 b and so forth may be formed of any relatively inertmetal provided that it is not cytotoxic. Metals typically used as inertelectrodes for biological fluids are silver, gold, platinum and theother platinum-group metals. Unfortunately these are very costly, maypermit or even catalyze some electrochemical reactions at their surfaces(especially if minor impurities are present), and the products of suchreactions may be cytotoxic.

For this reason the group of so-called “self-protecting” metals, whichon contact with water or aqueous solutions form thin, continuous, highlyinsoluble and biologically inert surface oxide layers sealing the metalsurface away from further fluid contact, are preferred in thisinvention. This oxide forms the only contact between the electricalsignal delivery system and the culture medium. Such metals includeniobium, tantalum, titanium, zirconium, molybdenum, tungsten andvanadium. Aluminum and stainless steels share this property but to amuch lesser degree, since they are slowly attacked by solutionscontaining chloride ion (as nearly all biological fluids do).

Oxide formation on such a metal can be enhanced, and the oxide thicknessincreased in a closely controllable manner, through anodization. Uniformoxide thickness gives uniform capacitance per unit area of metalsurface, in turn yielding relatively uniform signal intensity over thesurface almost regardless of its shape in the fluid. Small breaks in theoxide, caused by cutting and forming, heal rapidly by further reactionwith the fluid.

Niobium is preferred especially for this application since, thanks tothe vivid and stable colors created by light interference in the surfaceoxide (Nb₂O₅) produced by anodization, it is popular in jewelry and thusavailable at reasonable cost in convenient forms and a variety of stockcolors. Rio Grande Jeweler's Supply, for example, stocks 20- and22-gauge round niobium wire pre-anodized to “purple,” “pink,” “darkblue,” “teal,” “green” and “gold,” each color representing a differentoxide thickness. The wire is easily worked and formed to any desiredelectrode shape. Given the refractive index of Nb₂O₅ (N_(D)=2.30) andits dielectric constant (ε_(R)=41 ε₀), the oxide thickness may bemeasured easily from the light reflection spectrum, and the resultingcapacitance per unit of area or wire length may be calculated. “Purple”wire has the thinnest oxide, measured at 48 nM from the 420-nM peakreflectance, and thus for 22-gauge “purple” wire (0.0644 cm diameter;Rio Grande catalog number 638-240) the capacitance was calculated at0.154 microfarad per centimeter of wire length. Direct measurementinitially gave much higher readings due to oxide breaks, but after 24hours in saline the measured capacitance had stabilized at 0.158microfarad per centimeter, close to the predicted value.

Bridges 112 a, 112 b and so forth thus function electrically much asconventional salt bridges do, save that there is no possibility of fluidor ion flow through them, thus avoiding possible cross-contaminationbetween chambers or between a chamber and an end well. In addition, theproblems of evaporation and possible breakage encountered withconventional salt bridges, and the inconvenience of working with agar orother gelling agents, are avoided. Since they are electricallycapacitive, the bridges block direct current and thus the signalreaching the chambers is charge-balanced between phases, with anydirect-current component removed.

All bridge ends making contact with the growth medium preferably havethe same approximate dimensions and contain roughly the same length ofwire, so all have roughly equal capacitance, and are placed against thenarrow ends of culture chambers which themselves are preferablyrectangular, as shown in the magnified chamber 110 b in FIG. 4. Thebridge ends making contact with the fluid in the end wells (for example,the left ends of bridges 112 a and 112 g in FIG. 4) may if desired begiven a different form to enhance contact, decrease capacitance, and/orbetter fit the size and shape of the end wells if these differ from theculture chambers. For example, when using round end wells the bridgeends immersed in them may conveniently be formed as spirals 144 as shownin FIG. 4.

In summary therefore, the biological systems as contemplated by thepresent invention comprise the following elements: electricalsimulators, anodized metal electrodes, and cells. Suitable PEMF signalsfor use in such systems include waveforms as described for example inFIGS. 2 or 3. Practical applications of such signals include increasingproliferation, differentiation or mineralization of bone tissue,increasing BMP expression, or increasing nitric oxide production.

Tissue Engineering

The methods of the present invention may also be used in tissueengineering applications. Cells may be cultured using the methods andculture systems of the present invention in combination withbiologically compatible scaffolds to generate functional tissues invitro or ex vivo or transplanted to form functional tissues in vivo.Transplanted or host stem cells may also be selectively transplanted orattracted to a site of injury or disease and then stimulated with theelectrical signals described herein to provide enhanced healing orrecovery. Tissue scaffolds may be formed from biocompatible naturalpolymers, synthetic polymers, or combinations thereof, into a non-wovenopen celled matrix having a substantially open architecture, whichprovides sufficient space for cell infiltration in culture or in vivowhile maintaining sufficient mechanical strength to withstand thecontractile, compressive or tensile forces exerted by cells growingwithin the scaffold during integration of the scaffold into a targetsite within a host. Tissue scaffolds may be rigid structures forgenerating solid three-dimensional structures with a defined shape oralternatively, scaffolds may be semi-solid matrices for generatingflexible tissues.

The methods and culture systems of the present invention include the usescaffolds made from polymers alone, copolymers, or blends thereof. Thepolymers may be biodegradable or biostable or combinations thereof. Asused herein, “biodegradable” materials are those which contain bondsthat may be cleaved under physiological conditions, including enzymaticor hydrolytic scission of the chemical bonds.

Suitable natural polymers include, but are not limited to,polysaccharides such as alginate, cellulose, dextran, pullane,polyhyaluronic acid, chitin, poly(3-hydroxyalkanoate),poly(3-hydroxyoctanoate) and poly(3-hydroxyfatty acid). Alsocontemplated within the invention are chemical derivatives of saidnatural polymers including substitutions and/or additions of chemicalgroups such as alkyl, alkylene, hydroxylations, oxidations, as well asother modifications familiar to those skilled in the art. The naturalpolymers may also be selected from proteins such as collagen, zein,casein, gelatin, gluten and serum albumen. Suitable synthetic polymersinclude, but are not limited to, polyphosphazenes, poly(vinyl alcohols),polyamides, polyester amides, poly(amino acids), polyanhydrides,polycarbonates, polyacrylates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terephthalates, polyortho esters,polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyesters,polylactides, polyglyxolides, polysiloxanes, polycaprolactones,polyhydroxybutrates, polyurethanes, styrene isobutyl styrene blockpolymer (SIBS), and copolymers and combinations thereof.

Biodegradable synthetic polymers are preferred and include, but are notlimited to, poly α-hydroxy acids such as poly L-lactic acid (PLA),polyglycolic acid (PGA) and copolymers thereof (i.e., poly D,L-lacticco-glycolic acid (PLGA)), and hyaluronic acid. Poly α-hydroxy acids areapproved by the FDA for human clinical use. It should be noted thatcertain polymers, including the polysaccharides and hyaluronic acid, arewater soluble. When using water soluble polymers it is important torender these polymers partially water insoluble by chemicalmodification, for example, by use of a cross linker.

One of the advantages of a biodegradable polymeric matrix is thatangiogenic and other bioactive compounds can be incorporated directlyinto the matrix so that they are slowly released as the matrix degradesin vivo. As the cell-polymer structure is vascularized and the structuredegrades, the cells will differentiate according to their inherentcharacteristics. Factors, including nutrients, growth factors, inducersof differentiation or de-differentiation (i.e., causing differentiatedcells to lose characteristics of differentiation and acquirecharacteristics such as proliferation and more general function),products of secretion, immunomodulators, inhibitors of inflammation,regression factors, biologically active compounds which enhance or allowingrowth of the lymphatic network or nerve fibers, hyaluronic acid, anddrugs, which are known to those skilled in the art and commerciallyavailable with instructions as to what constitutes an effective amount,from suppliers such as Collaborative Research, Sigma Chemical Co.,vascular growth factors such as vascular endothelial growth factor(VEGF), EGF, and HB-EGF, could be incorporated into the matrix orprovided in conjunction with the matrix. Similarly, polymers containingpeptides such as the attachment peptide RGD (Arg-Gly-AsP) can besynthesized for use in forming matrices.

Kits

Kits are also provided in the present invention that combine electricalstimulators with biologically compatible scaffolds to support the growthand integration of cells into a unified tissue. Containers with built inelectrodes may be provided with the kit and the electrodes may be madeof a self-passivating material or other conventional electrodematerials. These kits may optionally include reagents such as growthmedia, and growth factors to promote integration of the cells with thescaffolds. Scaffolds included in the kit may be designed to havegrowth-promoting and adhesion molecules fixed to their surface. Suchkits are optionally packaged together with instructions on proper useand optimization.

Cells may be provided with the kit in a preserved form with a protectivematerial until such time that the cells are combined with other elementsof the kit to produce an appropriate tissue. In one embodiment, cellsare provided that are cryopreserved in liquid nitrogen or dessicated inthe presence of a compound such as trehalose. Cells may beundifferentiated progenitor cells, including stem cells; pluripotentstem cells, multipotent stem cells or committed progenitors.Alternatively, terminally differentiated cells may also be used withthese kits. Such kits may be designed to produce replacement tissue foruse in any organ system such as, but not limited to bone, cartilage,muscle, kidney, liver, nervous system, lung, heart, vascular system etc.

Cells may also be harvested from a patient in need of treatment toengineer replacement tissue from the patient's own tissue. Use of thepatient's own tissue provides a way to produce transplantation tissuewith reduced complications associated with tissue rejection.

In addition to purely electrical stimulation, a combination ofelectrical and mechanical stimulation in vitro may be found beneficialfor some purposes. Mechanical stimulation may consist of tensileloading, compressive loading, or shear loading. Typical setups are shownin cross-section in FIGS. 8 a through 8 e.

In each case of loading, the test setup is built around a culture wellor chamber 200 of any type familiar in the art, containing medium 202and a layer of cells 204 typically attached to a bottom sheet ormembrane 206 which may or not be a part of the rigid mechanical bottom208 of the culture well. Electrodes 210, of any useable metal asdescribed inter alia but preferably of a self-protecting metal and morepreferably of anodized niobium, are placed in chamber 200 in such a wayas to create relatively uniform current distribution throughout medium202.

For tensile loading, membrane 206 forms an additional or “false” bottomin culture well or chamber 200 as shown in FIG. 8 a. Membrane 206 may bemade from any suitably flexible and elastic material to which the cellswill attach themselves, such as silicone rubber which has been plasmaetched. Tube 212 connects space 214 between membrane 206 and rigidchamber bottom 208 with an external pump or other source of steady orfluctuating pressure or vacuum 216. The intermittent operation ofpressure or vacuum source 216 causes membrane 206 to flex up and down,creating intermittent tension in the membrane and thus in cell layer 204attached to it. Alternatively, source 216 may apply little or nopressure across membrane 206 for an extended period, allowing cells 204to colonize the membrane in its unstretched state, then apply adifferent pressure thereby stretching membrane 206, for example at apoint in culture growth at which cells 204 have just reached confluenceand established gap-junction contact.

For compressive loading, culture well or chamber 200 is instead sealedwith a cover 220 and connected to pressure source 216 directly as shownin FIG. 8 b. Source 216 creates a steady or fluctuating hydrostaticpressure in medium 202 which is thus applied directly to cell layer 204.

As an alternative means for compressive loading, tube 212 and pressuresource 216 are eliminated and chamber cover 220 takes the form of amovable piston through which steady or fluctuating pressure may beapplied directly to medium 202 and thus to cells 204, as shown in FIG. 8c.

For shear loading, culture well 200 is connected to pressure source 216instead via two tubes 212 a and 212 b through which medium 202 iscirculated, as shown in FIG. 8 d. This flow may be either constant in asingle direction, intermittent, or oscillatory. Each tube is preferablyequipped with baffles 220 to achieve more uniform flow, as generallyindicated by arrow 222. Baffles 220 may be made separate from electrodes210 as shown, or alternatively the electrodes may be perforated orotherwise made discontinuous so as themselves to form baffles. Themotion of medium 202 and its friction against cell layer 204 generatethe desired shear loading.

As an alternative means for providing shear loading, tubes 212 a and 212b and pressure source 216 are replaced with a moving impeller 230 whichmaintains medium 202 in motion relative to cell layer 204 as generallyindicated by arrow 232. Impeller 230 may take any of several forms, butmay advantageously be of cylindrical form as shown in FIG. 8 e, wherethe rigid bottom 208 of chamber 200 approximates the same form andmaintains a relatively uniform clearance from the impeller surface.Medium 202 is thereby swept continuously and at a steady speed overcells 204 simply by maintaining impeller 230 in rotation at a constantspeed. Alternatively, changing the speed of impeller 230 will change theflow velocity and thus the level of shear loading. Electrodes 210 arenot shown since they may take a variety of positions in thisarrangement. Preferably, however, rigid cell floor 208 and impeller 230are themselves made of suitable electrode metals, more preferably ofself-protecting metals and most preferably of anodized niobium, andthemselves function as the electrodes.

Differential Modulation of Bone Growth

The waveforms of the present invention as described above are alsouseful in methods for promoting the growth and repair of bone tissue invivo. As described above, stimulation with A-type waveforms promotesproliferation of cells. A-type waveforms also result in an increase inbone morphogenic proteins to promote differentiation. In one embodiment,an increase in BMP-2 and BMP-7 production is effected using A-type or toa lesser degree, B-type electrical signals. This effect is highlyvaluable and provides a method for enhancing the generation ofsufficient tissue for proper tissue healing in vivo, or to creatingtissue grafts. This signal is also valuable for providing sufficientcell mass for infiltration into a polymer scaffold for tissueengineering purposes. In another embodiment, as demonstrated by in vitrotesting, stimulation in vivo provides proliferation and differentiationof osteoblasts to increase the number of osteoblasts for mineralization.Such an increase in number of cells provides a method for filling ingaps or holes in developing or regenerating bone through electricalstimulation. Cells generated through proliferation induced by A-typewaveforms may be used immediately, or preserved using conventional cellpreservation methods until a future need arises.

Stimulation with B-type waveforms promotes proliferation to a smalldegree, and has actions different than A-type waveforms. Actionspromoted by B-type waveforms include, but are not limited tomineralization, extracellular protein production, and matrixorganization. The actions of B-type waveforms are also valuable andprovide methods to enhance the mineralization step and ossification ofnew bone tissue. In one embodiment, developing or regenerating bonetissue is stimulated with B-type waveforms to enhance the rate ofmineralization. It has been proposed that B-type waveforms may actthrough calcium/calmodulin pathways and also by stimulation of G-proteincoupled receptors or mechanoreceptors on bone cells. (Bowler, FrontBiosci, 1998, 3:d769-780; Baribault et al., Mol Cell Biol, 2006,26(2):709-717). As such, methods are also provided to modulate theactivity of calcium/calmodulin-mediated actions as well as G proteincoupled receptors and mechanoreceptors using electrical stimulation.Modulation of these cellular pathways and receptors are valuable topromote the growth and repair of bone tissue in vitro or in vivo.

Stimulation with C-type waveforms promotes bone regeneration, maturationand calcification. These waveforms are also valuable and provide methodsto enhance the mineralization step and ossification of new bone tissue.

Stimulation using D-type waveforms promotes cartilage development andhealing and bone calcification, and is useful for treating or reversingosteoporosis and osteoarthritis. Applications of these waveforms includein vivo applications such as repairing damaged cartilage, increasingbone density in patients with osteoporosis as well as in vitroapplications relating to the tissue engineering of cartilage forexample.

Methods are also provided for combination or sequential use of thewaveforms described herein for the development of a treatment regime toeffect specific biological results on developing or regeneratingosteochondral tissue.

In one embodiment, fractures in patients with a bone disorder may betreated with signals to heal fractures and then strengthen the bone. Asa non-limiting example of this embodiment, an osteoporotic patient witha fracture may be treated by first stimulating with an A-type signal topromote proliferation and release of growth factors and then a B-typewaveform to promote an increase in bone density at the site of repair toincrease bone mass density and prevent refracture.

In another embodiment, combining two or more types of waveformsdescribed herein may be used to promote the sequential proliferation,differentiation and mineralization of osteochondral tissues. As anon-limiting example of this embodiment, a culture of osteoblasts may begrown under the influence of a A-type signal in connection with or priorto connection with a polymeric matrix. After seeding the polymericmatrix, B-type signals are then administered to the cell-matrixconstruct to promote mineralization of a construct useful as a bonegraft.

In a third embodiment, two or more signals may be administeredsimultaneously to promote concomitant proliferation, differentiation andmineralization of osteochondral tissue in vivo or in vitro. Differentsignals may also be applied sequentially to osteochondral tissue inorder to yield a greater effect than delivering either signal alone. Thesequential process may be repeated as needed to produce additionaltissue (such as bone) by cycling through the two-step process enoughtimes to obtain the desired biological effect. As a specificnon-limiting example, A-type signals may be applied first to producemore bone cells by proliferation and then B-type signals may be appliedto induce the larger number of bone cells to produce more bone tissue(matrix, mineral and organization) and then repeated if needed. Theamount of bone produced using repetition of a sequential stimulationprotocol would be greater than that produced by either signal alone orin combination.

Progenitor Cell Stimulation

The methods and waveforms described herein may be applied toundifferentiated precursor cells to promote proliferation and/ordifferentiation into committed lineages. Such progenitor cells mayinclude, but are not limited to, stem cells, uncommitted progenitors,committed progenitor cells, multipotent progenitors, pluripotentprogenitors or cells at other stages of differentiation. Also includedare specifically osteoblasts and chondroblasts. In one embodiment,multipotent adult stem cells (mesenchymal stem cells or bone marrow stemcells) are stimulated with A-type signals in vitro to promoteproliferation and differentiation of the multipotent adult stem cellsinto specific pathways such as bone, connective tissues, fat etc.Combination or sequential administration with both signals is alsocontemplated for progenitor cell stimulation as previously described.

Alternatively, the waveforms and methods described herein may also beapplied to multipotent adult stem cells (mesenchymal stem cells or bonemarrow stem cells) in vivo to stimulate cells with A-type signals topromote proliferation and differentiation of the multipotent adult stemcells into specific pathways such as bone, connective tissues, fat etc.Combination or sequential administration with both signals is alsocontemplated.

Electrical stimulation of progenitor cells may also be accompanied byproliferation and differentiation factors known to promote proliferationor differentiation of progenitor cells. Proliferation factors includeany compound with mitogenic actions on cells. Such proliferation factorsmay include, but are not limited to bFGF, EGF, granulocyte-colonystimulating factor, IGF-I, and the like. Differentiation factors includeany compound with differentiating actions on cells. Such differentiationfactors may include, but are not limited to retinoic acid, BMP-2, BMP-7and the like.

The electrical waveforms described herein provide differential andcombination modulation on the growth and development of osteochondraltissue in vitro or in vivo. Increasing the proliferation of cells withA-type signals before mineralization increases the number of bone cellsand therefore provides an increase in the subsequent mineralizationeffected by stimulation with B-type signals. The waveforms of thepresent invention also promote proliferation and differentiation ofprogenitor cells through the release of nitric oxide and bonemorphogenic proteins.

Capacitive Coupling

Stimulation of in vitro and in vivo preparations is often difficult withself-passivating metals because it is difficult to obtain electricalconnections between metals. The present invention provides methods ofobtaining the benefits of using self-passivating metal electrodeswithout problems associated with obtaining solid electrical connections.Capacitive coupling of these electrodes provides a method to inducedirect current through the self-passivating metal electrodecircumventing the need for any electrical connection. In this methodelectrodes made from self-passivating metals such as niobium, tantalum,titanium, zirconium, molybdenum, tungsten and vanadium, aluminum andstainless steels are sterilized and placed in close proximity to apopulation of cells to be stimulated. Circuit wires are placed withinclose proximity to the metal electrodes in a conductive medium such assaline solution and electrical signals are transmitted through thecircuit wires with current being capacitively coupled from the wirethrough the saline and the oxide layer into the self-passivating metalelectrode to thereby stimulate the cell population. In one embodiment,capacitive coupling stimulation is used for in vitro applications suchas, but not limited to, cell culture. One culture dish may be stimulatedusing this method or several culture dishes or wells may be linkedtogether for uniform electrical stimulation.

In another embodiment, capacitive coupling stimulation is used for invivo applications where a sterile anodized metal electrode is implantedinto a patient in need of treatment and the circuit wires are placedoutside the patient in contact with the skin to induce a current in theimplanted metal electrode for an effective amount of time to promoterepair or growth of a tissue. For example, the outer end of theelectrode may form a flat coil just beneath the skin and the signal maybe coupled into it using a conventional skin contact electrode, placedon the skin directly over this coil. Portions of the capacitivleycoupled electrode from which close capacitive coupling to tissues is notdesired may be covered with any insulating material suitable for use inimplanted circuits, as is well known in the art, thus minimizing signalloss and undesired stimulation of tissues not being treated. In aspecific example such as bone repair, a sterile anodized metal electrodemade from a self-passivating metal is implanted into a patient in needof treatment and stimulated. After a sufficient period of time forrepair of the bone, the electrode may be removed from the patient.

Increase BMP Expression

The present invention further includes methods and apparatuses that useA-type and B-type waveforms for promoting the expression and release ofbone morphogenic proteins (BMPs) from stimulated cells. The electricalsignals described herein may be used to cause the release of BMPs atlevels sufficient to induce a benefit to the tissues exposed to thesignals. Benefit may occur in tissues not directly exposed to thesignals.

BMPs are polypeptides involved in osteoinduction. They are members ofthe transforming growth factor-beta superfamily with the exception ofthe BMP-1. At least 20 BMPs have been identified and studied to date,but only BMP 2, 4 and 7 have been able in vitro to stimulate the entireprocess of stem cell differentiation into osteoblastic mature cells.Current research is trying to develop methods to deliver BMPs fororthopedic tissue regeneration. (Seeherman, Cytokine Growth Factor Rev.June 2005;16(3):329-45). Methods are provided herein to induce therelease of BMPs in vitro or in vivo for orthopedic tissue regenerationthrough electrical stimulation instead of through delivery of exogenousBMPs in technically demanding and costly delivery methods.

In one embodiment, A-type and to a lesser degree, B-type waveforms areused to induce expression and release of endogenous BMPs. Release ofendogenous BMPs promotes the growth and differentiation of targettissues. Placement of stimulation electrodes provides a way to targetBMP expression to localized areas of an in vitro preparation or in vivoin a patient in need of increased BMP expression. In one embodiment,BMP-2 or BMP-7 or combinations thereof are released endogenously toeffect differentiation and growth of target tissue. In a specificembodiment, release of either or both of BMP-2 and BMP-7 promotesdifferentiation, mineralization, protein production and matrixorganization in bone or cartilage tissue.

Stimulation of Bone, Cartilage or Other Connective Tissue Cells byMitric Oxide

The methods and electrical signals described herein may also be used topromote repair and growth of bone, cartilage or other connectivetissues. In one embodiment, a B-type waveform increases the growth ofcells through the release of nitric oxide (NO). The waveforms may causethe release of nitric oxide at levels sufficient to induce a benefit tothe tissues exposed to the signals. Benefit may occur in tissues notdirectly exposed to the signals. Bone, cartilage, or other connectivetissue cell growth may be increased further by co-administration of anNO donor in combination with the electrical stimulation. NO donorsinclude but are not limited to sodium nitroprusside (SNP), SIN-1, SNAP,DEA/NO and SPER/NO. Bone, cartilage, or other connective tissue cellgrowth may be reduced by co-administering an NO synthase inhibitor incombination with the electrical stimulation. Such NO synthase inhibitorsinclude but are not limited to N(G)-nitro-1-arginine methyl ester(L-NAME), NG-monomethyl-L-arginine (L-NMMA), and 7-Nitroindazole (7-NI).Using these methods, bone, cartilage, or other connective tissue cellgrowth may be modulated depending on specific needs.

Application of the Apparatus and Methods of the Present Invention

By using the apparatus and methods of the present invention as describedherein, the apparatus and methods are effective in promoting the growth,differentiation, development and mineralization of osteochondral tissue.

The apparatus is believed to operate directly at the treatment site byenhancing the release of chemical factors such as cytokines which areinvolved in cellular responses to various physiological conditions. Thisresults in increased blood flow and inhibits further inflammation at thetreatment site, thereby enhancing the body's inherent healing processes.

The present invention is especially used in accelerating healing ofsimple or complex (multiple or comminuted) bone fractures including, butnot limited to, bones sawed or broken during surgery. The presentinvention can be used to promote fusion of vertebrae after spinal fusionsurgery.

The present invention may be used to treat nonunion fractures; treat,prevent or reverse osteoporosis; treat, prevent or reverse osteopenia;treat, prevent or reverse osteonecrosis; retard or reverse formation ofwoven bone (callus, bone spurs), retard or reverse bone calcium loss inprolonged bed rest, retard or reverse bone calcium loss in microgravity.In addition, the present invention may be used to increase local bloodcirculation, increase blood flow to areas of traumatic injury, increaseblood flow to areas of chronic skin ulcers and to modulate bloodclotting.

One of the areas where the present invention can also be used is toaccelerate the healing of damaged or torn cartilage. Also, the presentinvention can be used to accelerate the healing (epithelialization) ofskin wounds or ulcers.

The present invention may further be used to accelerate growth ofcultured cells or tissues, modulate cell proliferation, modulate celldifferentiation, modulate cell cycle progression, modulate theexpression of transforming growth factors, modulate the expression ofbone morphogenetic proteins, modulate the expression of cartilage growthfactors, modulate the expression of insulin-like growth factors,modulate the expression of fibroblast growth factors, modulate theexpression of tumor necrosis factors, modulate the expression ofinterleukines and modulate the expression of cytokines.

The methods and apparatuses of the present invention are furtherillustrated by the following non-limiting examples. Resort may be had tovarious other embodiments, modifications, and equivalents thereof which,after reading the description herein, may suggest themselves to thoseskilled in the art without departing from the spirit of the presentinvention and/or the scope of the appended claims.

EXAMPLES Example 1 Effect of PEMF Signal Configuration on Mineralizationand Morphology in a Primary Osteoblast Culture

The goal of this study was to compare two PEMF waveform configurationsdelivered with capacitative coupling by evaluating biochemical andmorphologic variations in a primary bone cell culture.

Methods

Osteoblast cell culture: Primary human osteoblasts (CAMBREX®,Walkersville, Md.) were expanded to 75% confluence, and plated at adensity of 50,000 cells/ml directly into the LAB-TEK™ (NALGE NUNCINTERNATIONAL™, Rochester, N.Y.) chambers described previously. Cultureswere supported initially with basic osteoblast media withoutdifferentiation factors. When the cultures reached 70% confluence withinthe chambers, media was supplemented withhydrocortisone-21-hemisuccinate (200 mM final concentration),β-glycerophosphate (10 mM final concentration), and ascorbic acid.Osteoblasts were incubated in humidified air at 37° C., 5% CO₂, 95% airfor up 21 days. Media was changed every two days for the course of theexperiment, 4 ml supplementing each chamber.

Electrical Stimulation: Cultures were stimulated for either 30 minutesor for 2 hours twice per day. Two electrical signal regimens wereselectively applied to the cells, one a continuous waveform indicated as“Signal A” (60/28 positive/negative signal duration in μsec), and theother a continuous waveform indicated as “Signal B” (200/28positive/negative signal duration in μsec). Intensity was measured insample runs as 2.4 mV/cm (peak to peak). Non-stimulated osteoblasts (NC)were plated at identical densities (as controls) in a similar manner.The following were measured using procedures in Detailed Methods:alkaline phosphatase, calcium, osteocalcin, and histology. Each of thefollowing graphs are keyed to the “A” signal, the “B” signal, 30-minutesduration as “1”, 2-hours duration as “2”, and NC (or confluence) as nocurrent (i.e. A1 would be A signal-30 minutes; B2 would be B-2 hours).

The electrical device used herein enables the application of continuouswaveform, electrical stimulation to multiple explants simultaneously.For each experiment, 6 pairs of explants were placed into individualwells in 4 ml of culture medium. Control specimens were cultured insimilar conditions, the only difference being the lack of signaldelivered. The present test configuration consisted of six test culturewells (17×42 mm) connected in series via a coiled section of niobiumwire.

Human osteoblast cells were established in LAB-TEK™ II slide wells(NALGE NUNC INTERNATIONAL™, Rochester, N.Y.), each with a surface areaof about 10 cm². Signals were applied to several chambers simultaneouslyby connecting them in serial via niobium wires which acted as a couplecapacitance. The stimulus was either a 9 msec burst of 200/28 μsecbipolar rectangular pulses repeating at 15/sec, delivering 9 mV/cm(similar to the standard clinical bone healing signal), designatedSignal B, or a 48 msec burst of 60/28 μsec essentially unipolar pulsesdelivering 4 mV/cm, designated Signal A. Cultures received either a30-minutes or a 2-hour stimulus twice a day. Samples were taken from themedia and analyzed at 7, 14, and 21 day time points for alkalinephosphatase, osteocalcin, matrix calcium and histology. Mineralizationaccompanying morphology was confirmed with Von Kossa stain. Allbiochemical analyses were performed by conventional assay techniques.

Results

PGE₂, production was assessed using commercially available ELISA kits(R&D SYSTEMS™, Minneapolis Minn.; INVITROGEN, INC.™, Carlsbad, Calif.).Results are expressed as pg/mg of tissue per 24 hours (μM/g/24 hrs).

Alkaline Phosphatase (AP): At the time points indicated in the studydesign, cells were lysed (Mammalian-PE, Genotech, St. Louis, Mo.) andthe supernatant collected. Alkaline phosphatase was measured by thecleavage of para-nitrophenyl phosphate (PNPP) to nitrophenyl (PNP) underbasic conditions in the presence of magnesium. The end product PNP iscolorimetric with an obsorption peak at 405 nanometers. Basic conditionswere achieved using 0.5 M carbonate buffer at pH 10.3. Culture media wasassayed directly for ALP activity. Cell layer ALP was extracted with asolution of triton X-100 and an aliquot measured for ALP activity.Alkaline Phosphatase was measured in both the supernatant and in themembrane following lysis buffer extraction (FIG. 5). As expected fromother studies (Lohman, 2003), alkaline phosphatase expression peakednear 7 days in the membrane. In the cells cultured under the “B”stimulus however, culture media continued to demonstrate an increase inmeasurable AP.

Osteocalcin: Osteocalcin (5800 daltons) is a specific product of theosteoblast. A small amount of osteocalcin is released directly into thecirculation; it is primarily deposited into the bone matrix. Studieshave shown that osteocalcin circulates both as the intact (1-49) proteinand as N-terminal fragments. The major N-terminal fragment is thepeptide (1-43). A Mid-Tact Osteocalcin Elisa Kit was selected for itshigh specificity. The assay is highly sensitive (0.5 ng/ml) and requiredonly a 25 microliter sample. Standards run simultaneously with ourexperimental groups offered a strong correlation to the expected valuesprovided by BTI manufacturers (BTI, Stoughton, Mass.). Osteocalcindeposition, measured subsequent to quenching the cultures and determinedfrom the matrix component, was more pronounced following the “B”stimulus and highest at 21 days (FIG. 6).

DNA content: Cell layer was extracted with 0.1 N sodium hydroxide and analiquot assayed for DNA content using CyQuant assay kit (INVITROGEN,INC.™, Carlsbad, Calif.). For cell samples extracted for ALP contentwith triton X-100 the extract was adjusted to 0.1N sodium hydroxideusing 1 N sodium hydroxide. Standard curves contain matching buffer. Forsamples also requiring protein content an aliquot was measured forprotein using dye binding method (Bradford).

Calcium: Calcium was determined by Schwarzenbach methodology witho-cresolphthalein complexone, which forms a violet colored complex. Byadding 2 ml of 0.5 M acetic acid overnight, calcium was dissolved andcontent was quantified against standards by colorimetric assay at 552 nm(CORE LABORATORY SUPPLIES™, Canton, Mich.).

Calcium Distribution in the culture was also assessed by histology.Cells were fixed in 2% glutaraldehyde, washed with cacodylate buffer,washed with PBS and then hydrated for staining as indicated. Each timeperiod was run in tandem; representative morphology is presented for 21days, comparing the “A” signal, with the “B” signal, and comparing bothsignals to the control (FIG. 6). For signal B, the most strikingobservation was in the distribution of the calcium with an apparentpreferential alignment that we interpreted as a “pseudo-cancellous”bone. For signal A, there appeared to be qualitatively more cellproliferation and less matrix production than signal B (however, signalA clearly had more matrix than with controls).

Osteometric analysis was developed and modified from the methodology ofCroucher. In this two dimensional chamber system, mean trabecular arearelative to total area of the grid sampled was studied. Using minimum of20 fields from two chambers at each intensity, the study examined boneformation, osteoid width, and cell number. Random specific grids weredeveloped for direct comparison and to remove bias. Additionally,osteoblast cultures in both stimulated and control chambers was staineddirectly by VonKossa method (Mallory, 1961) to examine histology andqualify the distribution of calcium within the cultures.

Conclusion

Alkaline phosphatase, which rose to a peak near the 10-14 day level andthen gradually subsided, was increased in the supernatant stimulated bySignal B. Osteocalcin deposition, measured subsequent to quenching thecultures and determined from the matrix component, was more pronouncedfollowing Signal B only and increased to its highest point at 21 days.Matrix calcium measured in mg/dl, and matrix calcium as a function ofthe area of the tissue culture plate were greatest with Signal B only.Mineral distribution as noted by histology and Von Kossa stainingvalidated the biochemical data from the assays. The B stimulus conferreda greater amount of mineral, and moreover suggested a reticulated2-dimensional pattern that may offer analogous tension dynamics as wouldbe expected in a 3-D trabecular array. Cell proliferation appearedqualitatively higher with Signal A vs control, whereas significantlyincreased mineralization and pattern was apparent at 21 days with SignalB.

That the two signal configuration produced very different effects isreadily explainable by a signal to noise ratio (SNR) analysis whichshowed the detectability of signal B was 10× higher than signal A,assuming a Ca/CaM target. This study demonstrates for the first timethat PEMF has the potential to effect structural changed resonant withtissue morphology. The geometric pattern apparent at 21 days of culture,mirrored the trabecular reticulation consonant with cancellous bone andstarkly contrasted the random orientation of the cells in both thecontrol and the cultures exposed to signal A at all time pointsevaluated. Such outcomes suggest that preferred signal configurationscan effect structural hierarchies that previously were confined totissue-level observations.

Example 2 Use of a Niobium “Salt” Bridge for In Vitro PEMF Stimulation

Introduction

A passive electrode system using anodized niobium wire was developed tocouple time-varying electric signals into culture chambers. The intentof the design was to reduce complexity and improve reproducibility byreplacing conventional electrolyte bridge technology for delivery ofPEMF-type signals, such as those induced in tissue by the EBI repetitivepulse burst bone grown stimulator, capacitively rather than inductively,in vitro for cellular, tissue studies. Anodized niobium wire is readilyavailable and requires only simple hand tools to form the electrodebridge. At usable frequencies, typically between 5 Hz and 3 MHz, DCcurrent passage is negligible.

Background

Capacitively-coupled electric fields have typically been introduced toculture media with conventional electrolyte salt bridges which havelimited frequency response and are difficult to use without risk ofcontamination for extended exposure times. Niobium (columbium) is one ofseveral metals which are self-passivating, forming thin but very durablesurface oxide layers when exposed to oxygen or moisture. Others aretantalum, titanium, and to a much lesser degree, stainless steels. Theprocess can be accelerated and controlled by anodization. A problem withself-passivation is that it makes reliable connection with other metalsdifficult. The present design avoids that difficulty.

Materials and Methods.

Niobium oxide, Nb₂O₅, is hard, transparent, electrically insulating andinert to water, common reagents and biological fluids over a wide pHrange. Anodizing niobium forms Nb₂O₅ with uniform thickness, showing arange of vivid light-interference colors valued for jewelry since no dyeis added, and yields stable and reproducible capacitances. Jeweler'sniobium is sold in standard colors each representing a different oxidethickness. Since the dielectric contact of Nb₂O₅ is unusually high(ε_(R)=41ε₀) and the layers are thin (48-70 nm), their capacitances aresurprisingly large. “Purple” niobium has the thinnest oxide and highestmeasures capacitance: 0.158 μF/cm for 22-gauge wire (Rio Grande#638-240), near the calculated value for 48 nM oxide (420 nM peakreflectance). In water or physiological salines, cut wire ends and smallflaws formed in bending quickly heal over with oxide, with no need forre-anodization.

The Niobium Bridge:

In this application niobium oxide forms the only electrical contact withthe medium and PEMF-type signals pass thought it capacitively. At signallevels below a few milliamperes, there is negligible electrolysis or pHchange to cause artifacts. Multiple chambers may be joined in series,each receiving identical signals. Each niobium bridge is bent forming asheet-like electrode at each end, with a typical capacitance of 0.56 μF.Placing electrode bridges at the ends of a rectangular chamber createsnearly uniform current distribution and voltage gradients throughout themedium. Gradients measured in a typical setup of culture changer,electrodes and PEMF-type signal as was previously shown in FIG. 4 anddescribed in the accompanying text, show a mean variation of ±3%, mainlynear electrodes or where the medium varies significantly in depth. Achamber or several joined in series are energized through specialniobium end bridges, each with its outer end coupled capacitivelythrough saline to a silver strip electrode forming a connectionterminal. This removes any need to connect niobium to itself or to anyother metal. Current is controlled by a series limiting resistanceR_(lim). The resulting bandpass (±3 dB of nominal) varies somewhat withR_(lim), but in a test setup ran from 5 Hz to 3 MHz, the highestfrequency tried. PEMF-type signals can thus be delivered undistorted invitro via capacitive coupling.

Experimental:

The utility of the niobium electrode bridge was tested on osteoblast andchondrocyte cultures using a B-type waveform as previously described.With this signal applied to OGM™ osteoblast medium (CAMBREX®,Walkersville, Md.) without cells present, the measured pH after 24 hourswas 8.29 compared with 8.27 in non-energized controls, suggestingnegligible electrolysis. Absence of physiologically significantcytotoxicity was shown by robust proliferation of osteoblasts,differentiation and development of a cancellous bone-like structure over21 days in OGM™ using both A-type and B-type waveforms, After 30 minuteand 2 hour exposure for 21 days to the waveform in culture, cells andmatrix were analyzed with energy-dispersive X-ray (EDX). No niobiumcould be detected. In other studies a B-type signal was applied to humancartilage cells (HCC) in culture medium containing 1% fetal calf serumfor 96 hours. The B-type signal caused a 154% increase in cell number asmeasure by DNA content of cell lawyer, again showing no significantcytotoxicity. In a direct comparison between the capacitively coupledsignal and an otherwise identical but electromagnetically coupledsignal, each delivered 30 minutes daily for four days, measuredincreases in osteoblast number by DNA differed significantly fromcontrols (157% for niobium, 164% for EM coupled) but not from eachother.

Conclusions.

A novel niobium electrode bridge has been developed to applycapacitively coupled PEMF-type signals to cells/tissues in culture. Thebandpass of the niobium bridge is 5 Hz to 3 MHz, so PEMF-type signalslike those used clinically for bone and wound repair pass withoutdistortion. Unlike standard electrolyte bridge configurations, theniobium bridge provides uniform current density within the culture dish.Application for extended PEMF exposures shows no electrolysis orphysiologically significant cytotoxicity.

Example 3 Stimulation of Cartilage Cells Using a Capactively CoupledPEMF Signal

Introduction

A PEMF signal similar to that used clinically for bone repair iscurrently being tested for its ability to reduce pain in joints ofarthritic patients. Of interest is whether this pain relief signal canalso improve the underlying problem of impaired cartilage.

Background

Compared to drug therapies and biologics, PEMF based therapeutics offera treatment that is easy to use, non-invasive, involves no foreign agentwith potential side effects, and has zero clearance time. Issues withPEMF therapeutics include identifying responsive cells, elucidating aphysical transduction site on a cell, and determining the biologicalmechanism of action that results in a cell response. The purpose of thisstudy was to determine whether a specific PEMF signal currently beingtested for pain relief (MEDRELIEF®, Healthonics, Inc, Ga.) couldstimulate cartilage cells in vitro and whether a biological mechanism ofaction could be unraveled.

Methods

Normal human cartilage cells (HCC; CAMBREX®, Walkersville, Md.) wereplated in rectangular cell chambers in monolayer. PEMF application wascapacitively coupled through a niobium electrode bridge system whichallowed a time varying current to flow uniformly through the chambers. Apulse-burst B-type signal as described herein is composed of a 10-msecburst of asymmetric rectangular pulses, 200/28 microseconds in width,repeated at 15 Hz. The PEMF signal was applied for 30 minutes pertreatment. Cell growth was assessed by DNA content of the cell layer.Nitric oxide (NO) content of culture media was assessed by the Griessreaction using an assay kit from INVITROGEN INC.® (Carlsbad, Calif.).Results are expressed as micromoles of NO per cell number as assessed byDNA content of the cell layer.

Results

A PEMF signal applied at 400 micro-amperes, peak-to-peak, to HCC cellsgrown in cultured media containing 1% fetal calf serum, every 12 hoursover a 96 hour period resulted in increased cell growth of 153±22%,p<0.001. Of interest was conditioned culture media collected 24 hoursafter the first PEMF treatment shows and increase in NO of 196±14%,p<0.001 which declined to non-significant levels at 96 hours. Undersimilar conditions when SNP (an NO donor-sodium nitroprusside) was addedto a final concentration of 3 micrograms/ml there was also an increasein NO at 24 hours (174±26%, p<0.001) and an increase in cell number at96 hours (168±22%, p<0.001) compared to non-treated controls. In asubsequent experiment the serum concentration was reduced to 0.1%, thePEMF applied at 40 microAmps once every 24 hours, and measurements takenafter 72 hours. PEMF treatment increased NO content in conditionedculture media to 154±30%, <0.01. As shown in FIG. 7, PEMF treatmentincreased cell number and this cell response was attenuated by L-NAME (anitric oxide synthase inhibitor).

Conclusion

These results suggest that a PEMF signal currently being tested toreduce joint pain due to arthritis may also provide a benefit tocartilage. The data indicates human cartilage cells can respond to thissignal with increased cell growth. Furthermore, a possible biologicmechanism of action for PEMF stimulated cartilage cell growth is throughrelease of NO. A similar response of cartilage calls to an NO-donorsupports this hypothesis. Although not conclusive, the data suggest thatincreased cell growth following PEMF treatment is either mediated by NO,or that NO is a required step in the mechanism for PEMF to produceincreased cell growth.

Example 4 PEMF Stimulation of BMP Production in a Primary OsteoblastCulture: Dependence on Signal Configuration and Exposure Duration

Introduction

As an adjunct to surgery in spine fusion, or for treatment ofrecalcitrant non-unions in long bones, PEMF has proven effective as anon-surgical therapeutic. Pilot work has demonstrated that osteoblastsrespond differently to both signal configuration and duration. One keydifference included a proclivity for depositing matrix in lieu of cellproliferation. Based on a proven efficacy of BMP in spine fusion and innon-unions, and on efforts demonstrating that BMP-2 and BMP-4 arestimulated by PEMF (Bodamyali, 1998), our study focused on betterunderstanding whether previous cell responses could be correlated withBMP regulation.

Objective

This study compared two PEMF waveform configurations delivered withcapacitive coupling, correlating biochemical and morphologic variationsin a primary bone cell culture with BMP regulation.

Methodology

Normal human osteoblast cells were established in 10 cm² individualculture chambers. Signals were applied to several chamberssimultaneously by connecting them in series via niobium wires whichacted as a coupling capacitance. Stimuli consisted of a continuous trainof either 60/28 microseconds rectangular, bipolar pulses designated as“signal A”, or 200/28 microsecond rectangular, bipolar pulses designatedas signal B, applying peak to peak electric fields of 1.2 mV/cm (in A)or 2.4 mV/cm (in B) uniformly to the cultures. Cultures were exposed for30 minutes (1), or 2 hours (2), twice a day, yielding groups A1, A2, B1and B2 for comparison. Aliquots previously used for membrane proteindeterminations were analyzed for BMP protein by ELISA assay, andmatrices previously used to determine calcium and interpret morphologywere used to isolate RNA that was subsequently analyzed by a two-stepreverse-transcriptase polymerase chain reaction (RT-PCR) using known andavailable sequence primers for (18s RNA) BMP-2 and BMP-7. Both thesignal that stimulated proliferation and that which stimulated matrixdeposition were analyzed for BMP regulation and protein translation.Samples from 7-, 14-, and 21-day time points were used to assureidentical comparisons for the assay.

Results

The chief outcomes of this experiment were sixfold; 1) BMP protein andmRNA for BMP were elevated in response to both stimuli, particularlythat of the “A” signal; 2) the 30 minute stimulus delivered twice perday offered nearly 40-fold increase in BMP-2 expression at 21 dayscompared to the 2-hour treatment, with the majority of the gain achievedduring the period between 14-21 days; 3) the 30-minute stimulus for the“A” signal provided a 15-fold increase in BMP-7 expression, again almostentirely noted between the 14- and 21-day analyses; 4) only moderateincreases in either BMP-2 or BMP-7 were seen with respect to the “B”signal; 5) this study provides the first evidence that BMP-7 expressionis promoted by PEMF stimulation and 6) although the proliferationassessment was qualitative, the mitogenic nature of BMP deposition is inaccord with previously published work. Work evaluating PEMF on atransformed cell line for short periods of time suggests that neitherBMP-3 nor BMP-6 is stimulated (Yajima, 1996). We did not evaluate ourmodel with respect to these growth factors.

Conclusion

Given the body of work that has shown BMP-2 to have morphogenetic andmitogenic properties, the proliferation of the cells in response to the“A” signal is not surprising. That the two signal configurationsproduced very different effects is potentially explainable by a SNRanalysis that suggest the dose of signal “B” can be 10× higher thansignal “A” with the assumption of a Ca/CaM transduction pathway. Perhapsmore unexpected was the normalized BMP-2 and BMP-7 levels despite theexaggerated matrix deposition afforded by the “B” signal. Bone formationis acutely dependent on a balance of growth factor and microtopographyof the surface—in fact, the presence of a smooth surface overrides thecell response to BMP-2 and accentuates dystrophic mineralization. Giventhe high degree of matrix organization and deposition seen in responseto the “B” signal, BMP transduction in and of itself seems insufficientfor productive bone formation and may occur by a separate targetingmechanism.

Example 5

Case Study: Treatment of Osteoporosis with PEMF Stimulation

One osteoporotic individual (female, age 50, T=−3.092 at start) usedelectrical stimulation using Signal B (200/30) for 4-5 days a week for3-5 hours each day. The patient remained on the same medications,supplements and activity for a one year period. Follow up bone densityscanning at 6 months and 12 months, revealed a 16% and 29% increase inbone mass density.

1. A method for modulating development or repair of bone, cartilage orother connective tissue comprising stimulating a developing orregenerating tissue with an electrical signal wherein the electricalsignal comprises an A-type, B-type, C-type or D-type signal for a timeperiod sufficient to modulate the development or repair of the tissue.2. The method of claim 1 further comprising stimulating a developingwith a second electrical signal wherein the second electrical signalcomprises an A-type, B-type, C-type or D-type signal.
 3. The method ofclaim 2 wherein the A-type signal comprises a long component having a βlength and a short component having an α length.
 4. The method of claim2 wherein the A-type signal comprises a long component of about 60 μsecin duration and a short component of about 28 μsec in duration.
 5. Themethod of claim 2 wherein the B-type signal comprises a long componenthaving a γ length and a short having an α length.
 6. The method of claim2 wherein the B-type signal comprises a long component of about 200 μsecin duration and a short component of about 28 μsec in duration
 7. Themethod of claim 2 wherein the two electrical signals are administeredsimultaneously or sequentially to promote proliferation,differentiation, or matrix production of cells in the tissue.
 8. Themethod of claim 1 wherein the tissue comprises progenitor cells.
 9. Themethod of claim 8 wherein the progenitor cells are selected from stemcells, uncommitted progenitors, committed progenitors, multipotentprogenitor cells, or pluripotent progenitor cells.
 10. The method ofclaim 1 wherein the electrical stimulation increases the production ofBMP-2 or BMP-7.
 11. The method of claim 1 wherein the electricalstimulation modulates the production of nitric oxide.
 12. A method forproducing or repairing bone, cartilage, or connective tissue comprisingstimulating a developing or regenerating tissue with an electricalsignal wherein the electrical signal comprises an A-type, B-type, C-typeor D-type electrical signal.
 13. The method of claim 12 furthercomprising mechanical loading in combination with the electrical signal.14. The method of claim 12 wherein the electrical stimulation iseffected using a capacitively coupled electrode.
 15. A method forproducing or repairing bone, cartilage or other connective tissuecomprising stimulating the tissue with a first electrical signal tostimulate proliferation, differentiation or matrix production oftissue-forming cells followed by stimulating the developing tissue witha second electrical signal to stimulate the proliferation,differentiation or matrix/mineral production of the tissue-formingcells.
 16. The method of claim 15 wherein the first electrical signalcomprises a A-type signal and the second electrical signal comprises aB-type signal.
 17. The method of claim 15 wherein the A-type signalcomprises a long component having a β length and a short componenthaving an α length.
 18. The method of claim 15 wherein the A-type signalcomprises a long component of about 60 μsec in duration and a shortcomponent of about 28 μsec in duration.
 19. The method of claim 15wherein the B-type signal comprises a long component having a γ lengthand a short having an α length.
 20. The method of claim 15 wherein theB-type signal comprises a long component of about 200 μsec in durationand a short component of about 28 μsec in duration.
 21. A capacitivelycoupled bridge electrode comprising an electrode made of an anodizedmetal.
 22. The electrode of claim 21 wherein the anodized metal isniobium, tantalum, titanium, zirconium, molybdenum, tungsten, vanadium,aluminum or stainless steels.
 23. A system comprising cells and anelectrical stimulator delivering a A-type signal or a B-type signal tothe cells.
 24. The system of claim 23 further comprising means formechanical loading of the cells.
 25. The system of claim 23 wherein theA-type signal comprises a long component between about 20 μsec and 100μsec in duration and a short component between about 5 μsec and 95 μsecin duration.
 26. The system of claim 23 wherein the A-type signalcomprises a long component of about 60 μsec in duration and a shortcomponent of about 28 μsec in duration.
 27. The system of claim 23wherein the B-type signal comprises a long component between about 100μsec and 1000 μsec in duration and a short component between about 5μsec and 75 μsec in duration.
 28. The system of claim 23 wherein theB-type signal comprises a long component of about 200 μsec in durationand a short component of about 28 μsec in duration.
 29. The system ofclaim 23 wherein the cells are selected from stem cells, uncommittedprogenitors, committed progenitor cells, multipotent progenitors,pluripotent progenitors or cells at other stages of differentiation. 30.The culture system of claim 23 further comprising a capacitively coupledbridge electrode made of an anodized metal.
 31. A kit for preparing atissue suitable for transplantation comprising cells and an electricalstimulator providing an electrical stimulus waveform wherein theelectrical stimulus waveform comprises a A-type, B-type, C-type orD-type signal wherein the waveform promotes proliferation,differentiation, or matrix production of the cells into a tissuesuitable for transplantation.
 32. The kit of claim 31 further comprisinga biodegradable or biostable scaffold.
 33. The kit of claim 32 whereinthe scaffold is made from a material selected from natural or syntheticpolymers.
 34. The kit of claim 32 wherein the scaffold is in associationwith growth-promoting or adhesion-promoting molecules.
 35. The kit ofclaim 31 further comprising means for mechanical loading of the cells.